Project Summary. To survive within the hostile environment of the infected host, pathogenic microorganisms must be able to sense and adapt to changing environmental conditions. This adaptation requires a coordinated regulation of multiple factors for growth and survival. Like many infectious agents, the human fungal pathogen Cryptococcus neoformans uses the conserved signaling molecule cyclic AMP (cAMP) to regulate its response to external stresses. The central components of cAMP signal transduction pathways are highly conserved among microorganisms. However, the main hypothesis of this proposal is that the upstream activating signals and the downstream effectors of cAMP are functionally specialized in microbial pathogens. This specialization allows pathogenic organisms to use cAMP signaling to control their virulence potential. Increased cAMP production activates a series of protein interactions that allows C. neoformans to adapt to the host environment. Specifically, the C. neoformans cAMP pathway regulates the induction of capsule and melanin, two cellular factors required for pathogenesis. This regulation occurs at the level of transcription. Therefore, the experiments of this proposal will identify trans- and cis-acting regulatory elements that mediate the effect of cAMP on the transcription of capsule-associated genes. Moreover, defining the regulatory networks controlled by cAMP-dependent transcription factors will allow us to explore broad issues in environmental sensing, cellular stress, and microbial virulence. Specific Aim 1 proposes bioinformatic and transcriptional profiling approaches to identify transcription factors that control C. neoformans capsule gene expression. Specific Aim 2 outlines detailed testing of selected transcriptional regulators to define their role in capsule induction. Experiments in Specific Aim 3 will define the direct and indirect target genes of the transcription factors identified in the first two Aims.